Article Figures & Data
Figures
- Figure 1. Long and short isoforms of CIP4 and FBP17 have opposing effects on cortical neuronal development.
(A) Schematics of long and short human isoforms of CIP4 and FBP17. The F-BAR/EFC domain is shown as a dimer and only one C-terminal half of the protein is shown for clarity. F-BAR, HR1, and SH3 regions are false-colored, space-filling diagrams based on the following PDB files: CIP4 F-BAR/EFC domain (2EFK), FBP17 F-BAR/EFC domain (2EFL), HR1 domains (2KE4), and SH3 domains (2CT4). (B) Images of living cortical neurons at 12 h postplating, cotransfected with mRuby-Lifeact (red) to label actin and EGFP-labeled F-BAR protein (green). Contrast on black and white images is inverted for clarity. (C) Images of fixed COS-7 cells transfected with different isoforms of CIP4 and FBP17 and labeled with phalloidin (f-actin) and DAPI (nuclei). (D–G) Box-and-whisker plots showing quantification of stage 1 neurons (with points showing data that falls outside of the 10–90 percentile) comparing the effects of the different isoforms on peripheral intensity (D), filopodia number (E), cell complexity (F), and tubule number (G). CIP4S-EGFP (n = 24 cells), CIP4L-EGFP (n = 30 cells), FBP17L-EGFP (n = 23 cells), or FBP17S-EGFP (n = 31 cells). (H) Stacked bar graph comparing the percentage of neurons in stage (st.) 1, 2, and 3 for neurons expressing EGFP (n = 58), CIP4S-EGFP (n = 72), FBP17L-EGFP (n = 75), or CIP4S-tdTomato and FBP17L-EGFP (n = 65) at 12 h postplating. Two-way ANOVA with Bonferroni post-test multiple comparison. (I) Image of a living cortical neuron cotransfected with CIP4S-Scarlet and FBP17L-EGFP. (J) Box-and-whisker plot showing average colocalization (Pearson’s correlation coefficient) of CIP4S and FBP17L in cortical neurons (n = 46 cells). (K) Co-IP with CIP4S-HA and either CIP4S-EGFP or FBP17L-EGFP in cortical neurons. Original blot was separated to show higher molecular weight proteins (CIP4S-EGFP and FBP17L-EGFP) and EGFP. This blot was reprobed with antibodies to HA and tubulin. (L) Quantification of three co-IPs with CIP4S-HA. One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images of neurons and 1 µm in insets; 15 μm in whole-cell images of COS-7 cells and 7 μm in insets.
Source data are available for this figure.
Source Data for Figure 1[LSA-2018-00288_SdataF1.jpg]
- Figure S1. CIP4 and FBP17 expression results in opposing phenotypes and do not associate with one another.
(A) Images of living cortical neurons cotransfected with mRuby-Lifeact (red) and either EGFP, CIP4S-EGFP, or FBP17L-EGFP (green). (B–F) Box-and-whisker plots showing quantification of stage 1 neurons comparing the effects of transfection of EGFP, CIP4S-EGFP, or FBP17L-EGFP on peripheral intensity (B), cell complexity (C), filopodia number (D), tubule number (E), and filopodial length (F) 12 h postplating. EGFP (n = 26 cells), CIP4S-EGFP (n = 24 cells), and FBP17L-EGFP (n = 24 cells). (G) Images of a cortical neuron cotransfected with CIP4L-EGFP and FBP17L-mRuby. (H) Box-and-whisker plot showing average colocalization (Pearson’s correlation coefficient) of CIP4L with FBP17L in cortical neurons (n = 28 cells). (I, J) Co-IPs from HEK-293 cells expressing either CIP4S-HA or FBP17L-HA and EGFP-labeled FBP17 and CIP4 constructs. (K) Images of CIP4 KO cortical neurons cotransfected with mRuby-Lifeact and either CIP4S-EGFP and FBP17L-EGFP. Data for all graphs are shown as box-and-whisker plots with data points showing data that falls outside of the 10–90 percentile. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm in insets.
- Figure 2. The localization and function of CIP4S and FBP17L is encoded by the C-terminal half of the protein, but not by the HR1 domain alone.
(A) Schematic of the C-terminal domain swaps CFLFFF and FCSCCC. Each letter represents a domain or region of the protein. (B) Images of living cortical neurons at 12 h postplating, cotransfected with mRuby-Lifeact, and EGFP-labeled protein or chimera. (C–F) Quantification of stage 1 neurons comparing the effects of the C-terminal swap constructs on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F). CIP4S-EGFP (n = 24 cells), CFLFFF-EGFP (n = 22 cells), FBP17L-EGFP (n = 24 cells), or FCSCCC-EGFP (n = 21 cells). (G) Stacked bar graph comparing the percentage of neurons in stage (st) 1, 2, and 3 for neurons expressing CIP4S-EGFP (n = 40) versus CFLFFF-EGFP (n = 45) and FBP17L-EGFP (n = 48) versus FCSCCC-EGFP (n = 41). Two-way ANOVA with Bonferroni post-test multiple comparison. (H) Schematic of the HR1 domain swaps CCSFCC and FFLCFF. (I) Images of living cortical neurons cotransfected with mRuby-Lifeact and EGFP-labeled protein or chimera. (J–M) Graphs showing quantification of stage 1 neurons comparing the effects of the HR1 domain swap constructs on peripheral intensity (J), filopodia number (K), cell complexity (L), and tubule number (M). CIP4S-EGFP (n = 24 cells), CCSFCC-EGFP (n = 22 cells), FBP17L-EGFP (n = 24 cells), or FFLCFF-EGFP (n = 23 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm in insets.
- Figure S2. The SH3 domain has little effect on localization and function of CIP4 and FBP17.
(A) Schematic representation of the SH3 domain swap chimeras CCSCCF and FFLFFC. (B) Images of cortical neurons cotransfected with mRuby-Lifeact and either CIP4S-EGFP, CCSCCF-EGFP, FBP17L-EGFP, or FFLFFC-EGFP. (C–F) Box-and-whisker plots showing quantification of stage 1 neurons comparing the effects of the SH3 domain swap constructs on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F). CIP4S-EGFP (n = 24 cells), CCSCCF-EGFP (n = 23 cells), FBP17L-EGFP (n = 24 cells), or FFLFFC-EGFP (n = 26 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. (G) Stacked bar graph comparing the percentage of neurons in stage 1, 2, and 3 for neurons expressing CIP4S-EGFP (n = 50) versus CCSCCF-EGFP (n = 49) and FBP17L-EGFP (n = 61) versus FFLFFC-EGFP (n = 51) at 12 h postplating. (H) Stacked bar graph comparing the percentage of neurons in stage (st) 1, 2, and 3 in the HR1 domain swap constructs for neurons expressing CIP4S-EGFP (n = 50), CCSFCC-EGFP (n = 53), FBP17L-EGFP (n = 61), and FFLCFF-EGFP (n = 57) at 12 h postplating. Two-way ANOVA with Bonferroni post-test multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm in insets.
- Figure S3. Active Cdc42 is necessary for tubule formation in stage 1 cortical neurons.
(A) Images of cortical neurons cotransfected with mRuby-Lifeact and FBP17L-EGFP (n = 22 cells) and either CA-Cdc42 (n = 24 cells) or DN-Cdc42 (n = 22 cells). (B) Box-and-whisker plot showing the average number of tubules in control conditions or with either CA-Cdc42 or DN-Cdc42. (C) Images of cortical neurons cotransfected with mRuby-Lifeact and FBP17L-EGFP, before and after treatment with the Cdc42 inhibitor ZCL278. (D) Box-and-whisker plot showing the percent change in number of tubules with Cdc42 inhibitor (ZCL278) treatment and after washout, relative to pretreatment levels (n = 7 cells). (E) Box-and-whisker plot showing the percent change in number of tubules with Rac1 inhibitor NSC23766 treatment and after washout, relative to pretreatment (n = 5 cells). Data for all graphs are shown as box-and-whisker plots with data points showing data that falls outside of the 10–90%. *P < 0.05 and ***P < 0.001, compared with the EGFP control (one-way ANOVA with Kruskal–Wallis post-test multiple comparisons). Scale bars represent 5 μm in whole-cell pictures and 1 μm in insets.
- Figure S4. The middle domains of CIP4S and FBP17L determine their localization and function in cortical neurons.
(A) Schematic representation of the middle domain swaps CFLFFC and FCSCCF. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and either CFLFFC-EGFP or FCSCCF-EGFP. (C–F) Graphs showing quantification of stage 1 neurons comparing the effects of middle domain swaps on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F). CIP4S-EGFP (n = 23 cells), CFLFFC-EGFP (n = 17 cells), FBP17L-EGFP (n = 24 cells), or FCSCCF-EGFP (n = 17 cells). (G) Stacked bar graph comparing the percentage of neurons in stage (st) 1, 2, and 3 for neurons expressing CIP4S-EGFP (n = 50) versus CFLFFC-EGFP (n = 54) and FBP17L-EGFP (n = 61) versus FCSCCF-EGFP (n = 52) at 12 h postplating. One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images of neurons and 1 µm in insets.
- Figure S5. The L1 region is necessary for membrane tubulation in COS-7 cells and cortical neurons.
(A) Images of COS-7 cells transfected with one of the following: C-CCC-EGFP, C----EGFP, CCS---EGFP, F-FFF-EGFP, F----EGFP, or FFL---EGFP, fixed and stained with phalloidin and DAPI. (B) Images of living CIP4 KO cortical neurons transfected with mRuby-Lifeact and either CFLCCC-EGFP or FCSFFF-EGFP. (C) Images of COS-7 cells transfected with either CCL---EGFP or FFS---EGFP, fixed, and stained with phalloidin and DAPI. (D) Images of cortical neurons transfected with mRuby-Lifeact and either CCL---EGFP or FFS---EGFP. (E) Schematic representation of Linker 2 deletion constructs of CIP4S-EGFP (CCSC-C) and FBP17L-EGFP (FFLF-F). Images of living cortical neurons transfected with mRuby-Lifeact and either CCSC-C-EGFP or FFLF-F-EGFP. Scale bars represent 5 µm in whole-cell images of neurons and 1 µm in insets and 15 μm in whole-cell images of COS-7 cells and 7 μm in insets.
- Figure 3. Swapping the first linker region of CIP4S and FBP17L reverses localization and function.
(A) Schematic of the L1 domain swaps CFLCCC and FCSFFF. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and EGFP-labeled protein or chimera at 12 h postplating. (C–F) Quantification of stage 1 neurons, comparing the effects of the L1 swap constructs on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F) at 12 h postplating. CIP4S-EGFP (n = 24 cells), CFLCCC-EGFP (n = 47 cells), FBP17L-EGFP (n = 23 cells), or FCSFFF-EGFP (n = 37 cells). (G) Stacked bar graph comparing the percentage of neurons in stage (st) 1, 2, and 3 for neurons expressing CIP4S-EGFP (n = 45) versus CFLCCC-EGFP (n = 72) and FBP17L-EGFP (n = 49) versus FCSFFF-EGFP (n = 68) at 12 h postplating. Two-way ANOVA with Bonferroni post-test multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bar represents 5 µm in whole-cell images and 1 µm in insets.
- Figure 4. The F-BAR and first linker region are required for membrane binding and bending.
(A) Schematic of deletion constructs of CIP4S and FBP17L. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and EGFP-labeled protein or deletion mutant at 12 h postplating. (C–F) Quantification of stage 1 neurons comparing the effects of the deletion constructs on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F) at 12 h postplating. CIP4S-EGFP (n = 24 cells), C-CCC-EGFP (n = 35 cells), C---- EGFP (n = 21 cells), CCS--- EGFP (n = 22 cells), FBP17L-EGFP (n = 23 cells), F-FFF EGFP (n = 33 cells), F---- EGFP (n = 28 cells), and FFL--- EGFP (n = 29 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm in insets.
- Figure 5. The CIP4 HR1 domain is required for peripheral localization.
(A) Schematic of the FFSCFF chimera. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and either EGFP-labeled protein or chimera at 12 h postplating. (C–F) Quantification of stage 1 neurons comparing the effects of chimeric constructs on peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F) at 12 h postplating. FBP17S (n = 31 cells), FFSCFF (n = 30 cells), CIP4S (n = 24 cells), and CFSCCC (n = 37 cells). (G) Schematic of the CFSCCC chimera. (H) Images of cortical neurons cotransfected with mRuby-Lifeact and either EGFP-labeled protein or chimera at 12 h postplating. (I) Images of cortical neurons cotransfected with mRuby-Lifeact and either EGFP-labeled protein or chimeric deletion at 12 h postplating. (J–M) Quantification of stage 1 neurons comparing the effects of chimeric deletion constructs on peripheral intensity (J), filopodia number (K), cell complexity (L), and tubule number (M) 12 h postplating. CIP4S (n = 16 cells), CCSC-- (n = 25 cells), FFSC-- (n = 20 cells), and FFSF-- (n = 17 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm in insets.
- Figure S6. Deletion of the L2 and SH3 domains affect the function of CIP4S and CIP4S-like chimeras.
(A–C) Images of cortical neurons transfected with CIP4S-EGFP, CCSC--EGFP, or FFSC--EGFP. (A′–C′) Kymographs taken from the blue-, yellow-, and red-colored lines in (A–C). Each kymograph represents the relative movement (distance = 5.9 μm) of the membrane over a 5-min time-lapse. Scale bar in (C) represents 5 µm. Black lines in (A–C) indicate additional points of kymograph measurement. (D) Quantification of the average number of protrusion events (five lines per cell) per cell. CIP4S-EGFP (n = 10 cells), CCSC--EGFP (n = 10 cells), and FFSC—EGFP (n = 7 cells). (E) Average length of protrusion per cell. (F) Images of cortical neurons transfected with mRuby-Lifeact and CCSCC-EGFP. (G–J) Graphs showing quantification of stage 1 neurons comparing the effects SH3 deletion and L2+SH3 deletion on peripheral intensity (G), filopodia number (H), cell complexity (I), and tubule number (J). CIP4S-EGFP (n = 16 cells), CCSCC-EGFP (n = 19 cells), and CCSC--EGFP (n = 25 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. (K) Stacked bar graphs comparing the percentage of neurons in stage (st) 1, 2, and 3 neurons expressing CIP4S-EGFP (n = 33), CCSCC- EGFP (n = 30), and CCSC--EGFP (n = 36) at 12 h postplating. Two-way ANOVA with Bonferroni post-test multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.
- Figure S7. The polybasic region within the first linker region of CIP4 is responsible for F-BAR-dependent membrane tubulation.
(A) Multiple sequence alignments of CIP4 and FBP17 were performed with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), followed by manual manipulation (accession numbers). CIP4Hs (NP_001275891.1), CIP4Pt (XP_016790317.1), CIP4Ms (NP_001229318.1), CIP4Dr (XP_017209563.1), TOCA1Ce (NP_741723.1), CIP4Dm (NP_001261412.1), FBP17Hs (AAI43514.1), FBP17 Pt (JAA43647.1), FBP17Rn (NP_620269.1), FBP17 Ms (NP_001171119.1), FBP17Xl (NP_001085826.1), FBP17Dr (NP_001116716.1), and TOCA2Ce (NP_499838.2). Basic residues within the PBR (blue box) are shown in blue and proline residues within the poly-PxxP region (red box) are shown in red. (B) Images of fixed COS-7 cells transfected with CIP4L-EGFP, CIP4L-7Q-EGFP, CCL---EGFP, and CCL---7Q-EGFP, CIP4S-EGFP, CIP4S-7Q-EGFP, CCS--- EGFP, and CCS---7Q-EGFP and labeled with phalloidin and DAPI. (C) Images of living cortical neurons transfected with mRuby-Lifeact and either CCL---EGFP, CCL---7Q-EGFP, CCS---EGFP, or CCS---7Q-EGFP. Scale bars represent 5 µm in whole-cell images of neurons and 1 µm in insets, and 15 μm in whole-cell images of COS-7 cells and 7 μm in insets.
- Figure 6. The PBR is required for membrane bending and the poly-PxxP region is required for tubulation in cortical neurons.
(A) Schematics of the CIP4 L1 PBR in the short and long isoforms, highlighting basic amino acids (K/R) in blue and the K/R-Q mutations in red. (B) Images of living cortical neurons cotransfected with mRuby-Lifeact and EGFP-labeled proteins or mutant proteins 12 h postplating. (C–F) Quantification of stage 1 neurons comparing the effects of the 7Q mutationson peripheral intensity (C), filopodia number (D), cell complexity (E), and tubule number (F) 12 h postplating. CIP4L-EGFP (n = 29 cells), CIP4L-7Q-EGFP (n = 28 cells), CIP4S-EGFP( = 24 cells), or CIP4S-7Q-EGFP (n = 34 cells). (G) Schematic of the L1L in CIP4L and FBP17L showing the PxxP motifs highlighted in blue and the AxxA mutations highlighted inred. (H) Images of living cortical neurons cotransfected with mRuby-Lifeact and either EGFP-labeled protein or mutant 12 h postplating. (I–L) Quantification of stage 1 neurons comparing the effects of the AxxA mutations on peripheral intensity (I), filopodia number (J), cell complexity (K), and tubule number (L) 12 h postplating. CIP4L-EGFP (n = 29 cells), CIP4L-AxxA-EGFP (n = 29 cells), CIP4S-EGFP (n = 24 cells), FBP17L-EGFP (n = 23 cells), FBP17L-AxxA-EGFP (n = 25 cells), or FBP17S-EGFP (n = 31 cells). One-way ANOVA with Kruskal–Wallis post-test multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant. Scale bars represent 5 µm in whole-cell images and 1 µm ininsets.
- Figure S8. CA Rac1 drives CIP4L, but not FBP17L, to the periphery.
(A) Images of primary cortical neurons transfected with CA Rac1-V12, mRuby-Lifeact, and either CIP4L-EGFP or FBP17L-EGFP. Note that a portion of CIP4L-EGFP is now localized at the periphery, similar to CIP4S. FBP17L-EGFP does not localize to the periphery, maintaining its localization to tubules. (B) COS-7 cells transfected with either CIP4L-EGFP or CIP4L-EGFP with CA-Rac1-V12 (unlabeled), fixed, and stained with phalloidin (for F-actin) and DAPI to stain nuclei. Note in neither of these experiments does CIP4L-EGFP localize to the periphery, as it does in primary cortical neurons. Scale bars represent 5 µm in whole-cell images and 1 µm in insets, and 15 μm in whole-cell images of COS-7 cells and 7 μm in insets.
- Figure 7. Working model for CIP4 and FBP17 membrane localization and function.
(A) Schematic showing the localization and function of CIP4S and FBP17L. High levels of CIP4S expression results in concentration at the peripheral membrane, increased lamellipodia and veil protrusion, and inhibition of neurite outgrowth. In contrast, high levels of FBP17L expression results in concentration on tubules and excessive tubulation, more prominent filopodia formation, and promotion of precocious neurite outgrowth. A wild-type cell is shown with endogenous levels of CIP4S and FBP17L between these two extremes. (B) A model describing the function of the PBR and HR1 domain in CIP4S and FBP17S. The F-BAR/EFC domain of CIP4 or FBP17 alone cannot bind or bend membrane. The F-BAR/EFC domains of CIP4 and FBP17 require the positive amino acid residues within the PBR to bind and tubulate membrane in primary cortical neurons (and COS-7 cells). The addition of the CIP4 HR1 domain relocates CIP4 to the peripheral plasma membrane, where it bends membrane but produces static (slowly extending/retracting) protrusions. Addition of the second linker region and SH3 domain results in dynamic (extending and retracting) protrusions. The addition of the FBP17 HR1 domain appears to prevent membrane bending, resulting in full-length FBP17S adopting a diffuse distribution, similar to EGFP. (C) A model describing the function of the poly-PxxP region in the L1L of CIP4L and FBP17L. Long isoforms of both CIP4 and FBP17, which contain multiple PxxP motifs (four in CIP4L and six in FBP17L), induce membrane tubulation in cortical neurons. When these poly-PxxP motifs are mutated to AxxA, the long isoforms are no longer able to form tubules and localize in a fashion similar to their short forms. As CIP4L contains the CIP4 HR1 domain, the AxxA mutation in CIP4L localizes to the peripheral membrane and causes lamellipodial/veil formation, whereas the AxxA mutation in FBP17L no longer tubulates, rather concentrating on vesicles.
Tables
- Table 1.
Summary of all proteins, chimeras, and point and deletion mutants used in this study.
Protein/chimera/mutant Nomenclature Localization Filopodia number Tubule number % Neurons in stage 1 CIP4S CCSCCC P <1 <1 90 CIP4S SH3 swap CCSCCF P <1 1–2 90 CIP4S + FBP17 L1S swap CFSCCC P <1 <1 ND FBP17 + CIP4 L1S/HR1/L2 swap FCSCCF P <1 <1 85 FBP17 C-terminal swap FCSCCC P 1–2 1–2 60 CIP4S ΔL2+SH3 CCSC- - P 1–2 1–2 55a CIP4S ΔSH3 CCSCC- P 0–1 1–2 55a FBP17S + CIP4 HR1 swap FFSCFF P 1–2 1–2 ND CIP4L PxxP region mutant CIP4L-AxxA P 1–2 2–4 ND FBP17S ΔL2+SH3 FFSC- - P ND ND ND FBP17 L1 swap FCSFFF P 1–2 <1 65 CIP4S HR1 swap CCSFCC P/C 2–4 <1 65 GFP - - - - - C 2–4 <1 60 FBP17S FFSFFF C 1–2 <1 ND CIP4S PBR mutant CIP4S-7Q C 1–2 <1 ND CIP4L PBR mutant CIP4L-7Q C 1–2 <1 ND CIP4 ΔL1 C-CCC C 1–2 <1 ND CIP4 F-BAR/EFC (1–300) C- - - - C 1–2 <1 ND CIP4 F-BAR/EFC + L1S(7Q) CCS- - -7Q C ND ND ND CIP4 F-BAR/EFC + L1L (7Q) CCL- - -7Q C ND ND ND FBP17 ΔL1 F-FFF C 1–2 <1 ND FBP17 F-BAR/EFC (1–300) F- - - - C 1–3 <1 ND FBP17L PxxP region mutant FBP17L-AxxA V/T 1–2 1–3 ND FBP17 F-BAR/EFC + L1S FFS- - - T ND ND ND CIP4 F-BAR/EFC + L1S CCS- - - T 2–3 3–6 ND CIP4L CCLCCC T 1–2 4–8 ND CIP4 F-BAR/EFC + L1L CCL- - - T ND ND ND FBP17 F-BAR/EFC + L1L FFL- - - T 2–3 4–8 ND CIP4 C-terminal swap CFLFFF T 2–4 4–8 50 CIP4 L1 swap CFLCCC T 2–4 6–12 45 CIP4 + FBP17 L1L/HR1/L2 swap CFLFFC T 1–3 6–12 40 FBP17L SH3 swap FFLFFC T 2–4 3–6 40 FBP17L HR1 swap FFLCFF T 2–4 4–8 45 FBP17L FFLFFF T 2–4 6–12 35 C, cytosol; ND, not determined; P, periphery; T, tubule; V, vesicle. Shading depicts the most CIP4S (orange) to the most FBP17L (blue) phenotype.
Staging determined at 12 h in vitro, Filopodia number is expressed per 10 µm of cell perimeter, and Tubule number is expressed per cell.
↵a In this set of experiments, only 75% of CIP4S neurons were in stage 1.
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